Use of isotope effects and pH studies to determine the chemical mechanism of Bacillus subtilis L-alanine dehydrogenase.
Analysis of deuterium isotope effects with L-alanine-d4 and L-serine-d3, and of pH profiles with the same substrates, shows that L-alanine is sticky (that is, reacts to give products 1-7 times as fast as it dissociates) while L-serin is not. The pH profiles show the following: (1) NH3 and monoanionic amino acids are the substrates; (2) a cationic acid group on the enzyme (probably lysine) with a pK of 9.0-9.6 in E-NAD, but a pK well above 10 in E-NADH, must be protonated for activity and good binding of inhibitors and is probably important for maintaining the proper conformation of the enzyme; (3) A cationic acid group on the enzyme (probably histidine) with a pK around 7 in both E-NAD and E-NADA must be unprotonated for oxidation of amino acids but protonated for binding and reaction of pyruvate. This latter group is the acid-base catalyst for the chemical reaction. In E-NAD, it is so positioned that it can hydrogen bond to (and thus when protonated enhance the binding of) a D-hydroxy or a carbonyl group of an inhibitor, but its state of protonation does not affect the binding of L-lactate or propionate. In E-NADH, it is so placed that it can hydrogen bond to both D- and L-hydroxy groups, as well as in carbonyl groups. A chemical mechanism is postulated in which the dehydrogenation of L-alanine by NAD to produce iminopyruvate is followed by attack of water from the same side from which the hydride was removed. The catalytic histidine transfers a proton from the attacking water to the amino group of the resulting carbinolamine and then removes a proton from the hydroxyl group of the carbinolamine as ammonia is eliminated to give pyruvate.
D000409:Alanine / Q000378:metabolism; D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003903:Deuterium / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008433:Mathematics; D009243:NAD / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D009243:NAD; D012694:Serine; D003903:Deuterium; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase; D000409:Alanine